p38 and class IA PI3K mediate neutrophil apoptosis by controlling ROS generation. (A) Inhibition of p38 or PI3Ks before TNF-α stimulation abolishes ROS generation. Time course of ROS generation after TNF-α and FAS antibody stimulation. Neutrophils were pretreated with vehicle or 100nM wortmannin, 10μM SB203580, or 20μM of the NADPH oxidase inhibitor DPI before TNF-α stimulation (50 ng/mL). Cells were also treated with anti-FAS antibody (n > 5). (B) Class IA PI3Ks control ROS generation after TNF-α stimulation. Neutrophils were treated with vehicle (Control, Medium), diverse class IA PI3K inhibitors (for concentrations, see Figure 3B), or 10μM of the pan-caspase inhibitor VAD before TNF-α stimulation (50 ng/mL) (n > 5, except pretreatment with AS604850, where n = 2). (C) Inhibition of PI3Ks does not affect the viability of neutrophils derived from CGD patients. Neutrophils from 3 different CGD patients (who lack a functional NADPH oxidase) were analyzed for ROS generation (left panel) and for viability (right panel; ethidium bromide staining and flow cytometry) after PI3K or NADPH oxidase inhibition and TNF-α stimulation.